Computational Design of Unnatural Amino Acid Dependent Metalloproteins

描述(由申请人提供)：非天然氨基酸的定点结合和计算蛋白质设计领域的融合代表着一条目前未被探索但很有希望的生化研究途径。虽然已经开发了针对自然产生的蛋白质的计算方法，但用这些技术处理非天然氨基酸的能力还有待充分探索。这项研究旨在开发一种计算方法，允许设计含有非天然氨基酸的蛋白质，最终目标是产生具有治疗潜力的新型非天然氨基酸依赖酶。华盛顿大学贝克实验室成员开发的Rosetta软件套件将首先用于设计铁结合蛋白质，这种蛋白质利用非天然的金属结合氨基酸联吡啶丙氨酸--最初是由舒尔茨和斯克里普斯研究所的同事加入蛋白质中的。由于这种非天然氨基酸对铁具有内在的亲和力，因此在蛋白质中设计金属结合部位的困难问题应该变得更容易计算。作为第二个目标，将同时设计一个多巴胺的结合部位(它将为铁提供两个氧配体)。像多巴胺这样的儿茶酚与生俱来就对铁有很高的亲和力，这表明经过改造的蛋白质可以作为这类重要小分子的传感器。最后，将对儿茶酚结合蛋白进行进一步的计算设计，目标是创建一种非天然氨基酸依赖的外源双加氧酶样酶。这种酶可对多氯联苯化合物等持久性拟人毒素的生物修复产生深远影响。使用舒尔茨实验室成员开发的技术，设计的含有非天然氨基酸的蛋白质将在细菌表达系统中生产。然后，将使用一系列生物分析技术对纯化的蛋白质进行分析，这些技术将检查金属或儿茶酚的结合能力，或根据特定目的检测酶的活性。在实验过程中收集的数据将用于未来设计本项目范围内和以外的其他含有非天然氨基酸的蛋白质。因此，这项研究应该在生物科学领域产生深远的影响，其范围将超出上述项目。由于这项建议中探索的两个科学领域目前都处于快速增长状态，在这项研究过程中收集的任何信息都将指导进一步的计算设计工作，涉及其他现有的、基因编码的非天然氨基酸，以及未来开发的那些氨基酸。 与公共健康相关：这项研究最终寻求改造含有非天然氨基酸的蛋白质，这些蛋白质具有催化降解多氯联苯环境毒素的能力。此外，在研究过程中开发的计算方法将提供重要信息，指导未来设计含有治疗和其他有用功能的非天然氨基酸的蛋白质。

DESCRIPTION (provided by applicant): The confluence of the fields of site-specific incorporation of unnatural amino acids and computational protein design represents a currently unexplored but promising avenue of biochemical research. While computational methods have been developed for naturally occurring proteins, the ability to treat non-natural amino acids with these techniques has yet to be fully explored. The research proposed seeks to develop a computational method that allows design of proteins containing unnatural amino acids, with the ultimate goal of generating novel unnatural amino acid dependent enzymes with therapeutic potential. The Rosetta suite of software developed by members of the Baker lab at the University of Washington will first be used to design iron binding proteins that utilize the metal binding unnatural amino acid bipyridyl alanine - first incorporated into proteins by Schultz and co-workers at The Scripps Research Institute. As this unnatural amino acid has inherent affinity for iron, the difficult problem of designing a metal binding site within a protein should be rendered more computationally tractable. As a second goal, a binding site for dopamine (which will provide two oxygen ligands for the iron) will be concurrently engineered. Catechols like dopamine have inherently high affinities for iron suggesting the engineered proteins could serve as sensors for this important class of small molecules. Finally, the catechol binding proteins will be further designed computationally with the goal of creating a non-natural amino acid dependent extradiol dioxygenase like enzyme. Such an enzyme could have a far-reaching impact with respect to bioremediation of persistent anthropomorphic toxins such as polychlorinated biphenyl compounds. The designed unnatural amino acid containing proteins will be produced in a bacterial expression system using techniques developed by members of the Schultz laboratory. Purified proteins will then be analyzed using a host of bioanalytical techniques that will examine metal or catechol binding abilities, or enzymatic activity depending on the specific aim. Data collected in the course of experimentation will be used for future design of other unnatural amino acid containing proteins both within the scope of this project, and beyond. Consequently, this research should have far reaching impacts within the biological sciences that will extend beyond the projects described above. As both of the scientific fields explored in this proposal are currently in a state of rapid growth, any information gleaned in the course of this research will guide further computational design efforts involving other currently available, genetically encoded non-natural amino acids, as well as those developed in the future. PUBLIC HEALTH RELEVANCE: This research ultimately seeks to engineer unnatural amino acid containing proteins that possess the ability to catalytically degrade polychlorinated biphenyl environmental toxins. Additionally, the computational methods developed in the course of the research will provide vital information that will guide future efforts for the design of unnatural amino acid containing proteins with therapeutic and other useful functions.